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WO2010126045A1 - Colorant cyanine à base de pyrazole contenant un cation ammonium quaternaire - Google Patents

Colorant cyanine à base de pyrazole contenant un cation ammonium quaternaire Download PDF

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Publication number
WO2010126045A1
WO2010126045A1 PCT/JP2010/057467 JP2010057467W WO2010126045A1 WO 2010126045 A1 WO2010126045 A1 WO 2010126045A1 JP 2010057467 W JP2010057467 W JP 2010057467W WO 2010126045 A1 WO2010126045 A1 WO 2010126045A1
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group
general formula
represented
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Japanese (ja)
Inventor
山本　直之
竜雄 黒澤
諭 挟場
睦廣 伊逹
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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Priority to US13/318,089 priority Critical patent/US8546586B2/en
Priority to ES10769743.5T priority patent/ES2508340T3/es
Priority to EP10769743.5A priority patent/EP2426123B1/fr
Priority to JP2011511413A priority patent/JP5598468B2/ja
Publication of WO2010126045A1 publication Critical patent/WO2010126045A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups

Definitions

  • the present invention relates to a novel quaternary ammonium cation-containing pyrazole cyanine dye.
  • cyanine dye derivatives are widely used as dyes having fluorescence characteristics in the near-infrared region of 500 nm or more.
  • Cy3, Cy5 manufactured by GE Healthcare Biosciences (formerly Amersham Biosciences)] are known as leading fluorescent reagents.
  • Cy3 and Cy5 have a structure in which two indolenine skeletons are linked by a polymethine chain (indolenine cyanine dye).
  • indolenine cyanine dye a polymethine chain
  • a basic skeleton for example, high water solubility, avoidance of association state, etc.
  • Various cyanine dye derivatives aimed at increasing sensitivity have been developed (see, for example, Patent Document 1, Patent Document 2, Patent Document 3 and the like).
  • other cyanine dye derivatives those having a structure in which an azaindolenine skeleton and a pyrazole skeleton are bound by a polymethine chain have been developed (see, for example, Patent Document 4 and Patent Document 5).
  • cyanine dye derivatives having a pyrazole skeleton and an indole skeleton have been developed as fluorescent dyes having a higher sensitivity and higher water solubility in a shorter wavelength region than conventional optical systems (Patent Document 9).
  • the fluorescent dye can also be used as an internal standard substance in electrophoresis.
  • sufficient fluorescent dyes having high fluorescence intensity and capable of controlling the migration speed have not yet been obtained, and further development is desired.
  • the present invention has been made in view of the situation as described above, and has a structure in which a pyrazole skeleton and an indole skeleton are bonded with a polymethine chain, which has high fluorescence intensity in a short wavelength region, and in the molecule having the structure. It is an object of the present invention to provide a novel cyanine dye derivative capable of controlling the migration speed by further introducing a quaternary ammonium cation.
  • the present invention is a compound represented by the following general formula [1] or a salt thereof.
  • R 1 to R 6 are each independently a group of the general formula [101]
  • R 101 to R 104 each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, m represents an integer of 2 to 6; and two of R 102 to R 104 Or a nitrogen atom to which they are bonded to each other to form a heterocyclic ammonium cation.
  • R 12 represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkali metal atom, an organic ammonium ion, an ammonium ion or an anion
  • R 12 represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkali metal atom, an organic ammonium ion, an ammonium ion or an anion
  • R 13 represents a hydrogen atom, an alkali metal atom, an organic ammonium ion, an ammonium ion, or an anion
  • R 7 to R 10 are each independently an alkyl group, an alkynyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylsulfonyl group, an arylsulfonyl group, a substituted amino group, or a group derived from a carboxylic acid represented by the general formula [2]
  • R 11 represents a hydrogen atom, an alkyl group, an alkynyl group or an aryl group
  • n is
  • the pyrazole cyanine dye of the present invention is a novel cyanine dye derivative having a structure in which a pyrazole skeleton and an indole skeleton are bonded by a polymethine chain, and a quaternary ammonium cation is introduced into the molecule having the structure.
  • the pyrazole cyanine dye of the present invention has a quaternary ammonium cation group represented by the general formula [101], that is, the cation group has an oxygen atom and a nitrogen atom having a loan pair.
  • the cation group has an oxygen atom and a nitrogen atom having a loan pair.
  • FIG. 1 is a diagram showing a method for preparing a DNA-labeled antibody in Example 1.
  • FIG. 3 is a diagram showing a layout of capillary chips in Example 1.
  • FIG. 3 is a diagram schematically showing the arrangement relationship between a sample for electrophoresis on a capillary chip and a test solution in Example 1. In the method of this invention, it is an electropherogram when AFP is measured. In the method of this invention, it is an electropherogram when PIVKA II is measured.
  • the alkyl group of the alkyl group represented by R 1 to R 6 and having the group represented by the general formula [101] as a substituent is linear, branched or cyclic. Any of these may be used, preferably a straight-chain one, usually having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 5 carbon atoms. Specific examples thereof include a methyl group and an ethyl group.
  • the alkyl group having 1 to 3 carbon atoms represented by R 101 to R 104 is linear or branched, preferably linear, and usually has 1 carbon. -3, preferably 1-2, and more preferably 1. Specific examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and the like. Among them, a methyl group is preferable.
  • R 101 in the general formula [101] a hydrogen atom is preferable.
  • R 101 to R 103 it is particularly preferable that one is a hydrogen atom and the other two are alkyl groups (particularly a methyl group is more preferable).
  • M is usually an integer of 2 to 6, preferably 2 to 4, more preferably 2.
  • heterocyclic ammonium cation formed by two or three of R 102 to R 104 and the nitrogen atom to which they are bonded examples include a pyridinium group and an N-alkylpyridinium group having 1 to 3 carbon atoms (for example, N- Methylpyridinium group, N-ethylpyridinium group, Nn-propylpyridinium group, N-isopropylpyridinium group, etc.).
  • the substituent in the alkyl group having the group represented by the general formula [101] as a substituent is preferably substituted with the carbon atom at the terminal of the alkyl group.
  • Preferred specific examples of the group represented by the general formula [101] include, for example, dimethylammonioethylcarbamoyl group, trimethylammonioethylcarbamoyl group, diethylammonioethylcarbamoyl group, triethylammonioethylcarbamoyl group, dimethylammoniopropylcarbamoyl group.
  • R 1 to R 6 in the general formula [1] usually 1 to 4, preferably 1 to 2, more preferably one is an alkyl group having a group represented by the general formula [101] as a substituent. . Among them, it is preferable that either R 1 or R 2 , and preferably R 1 is an alkyl group having a group represented by the general formula [101] as a substituent.
  • Examples of the alkyl group having an amide bond of an alkyl group which may have a sulfonic acid-derived group as a substituent include a substituent (that is, a carboxylic acid-derived group represented by the general formula [2] or the general formula [3] In which the alkyl chain of the unsubstituted alkyl group is substituted with 1 to 10, preferably 1 to 3, and more preferably 1 amide bond. Can be mentioned.
  • the alkyl group of the alkyl group which may be present may be linear, branched or cyclic, and usually includes those having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms.
  • a part of the hydrogen atoms in the alkyl group which may have an amide bond may be the substituent (a group derived from the carboxylic acid represented by the general formula [2] or the general formula [3] And those substituted with the carbon atom at the terminal of the alkyl group are preferred.
  • any one of R 1 and R 2 (preferably R 2 ), one of R 3 and R 4 , and any of R 5 and R 6 One of them is preferably an alkyl group having 1 to 5 carbon atoms having a sulfonic acid-derived group represented by the general formula [3] as a substituent.
  • examples of the alkali metal atom represented by R 12 and R 13 include a lithium atom, a sodium atom, a potassium atom, and a rubidium atom. Among them, a sodium atom or a potassium atom And particularly preferably a sodium atom.
  • R 1 to R 6 in the general formula [1] at least one (preferably R 1 ) is preferably an alkyl group having a group represented by the general formula [101] as a substituent.
  • the number of groups represented by the general formula [101] contained in the compound represented by the general formula [1] of the present invention is usually an integer of 1 to 6, preferably 1 to 4, and preferably an integer of 1 to 2, 1.
  • Examples of organic ammonium ions represented by R 12 and R 13 include trialkylammonium ions.
  • the trialkylammonium ion may be linear, branched or cyclic, and usually includes those having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms.
  • the alkyl group having 1 to 10 carbon atoms represented by R 12 may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 1 carbon atoms. 6, more preferably 1 to 3, specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl Group, n-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, neohexyl group, n-heptyl group, Isoheptyl group, sec-heptyl group, tert-heptyl group, neoh
  • Preferred examples of the group derived from the carboxylic acid represented by the general formula [2] include, for example, a carboxyl group (—COOH), an anion thereof [carboxylate (—COO ⁇ )], an alkali metal salt thereof (eg, lithium salt, sodium salt). Salt, potassium salt, rubidium salt, etc.), ammonium salt thereof, organic ammonium salt thereof (for example, trimethylammonium salt, triethylammonium salt, tripropylammonium salt, etc.) and the like.
  • the group derived from the sulfonic acid represented by the general formula [3] include, for example, a sulfo group (—SO 3 H), an anion thereof [sulfonate (—SO 3 ⁇ )], an alkali metal salt thereof (for example, a lithium salt) Sodium salts, potassium salts, rubidium salts, etc.), ammonium salts thereof, organic ammonium salts thereof (for example, trimethylammonium salts, triethylammonium salts, tripropylammonium salts, etc.).
  • a sulfo group —SO 3 H
  • an anion thereof for example, a lithium salt
  • ammonium salts thereof for example, organic ammonium salts thereof (for example, trimethylammonium salts, triethylammonium salts, tripropylammonium salts, etc.).
  • any one of R 1 and R 2 is, for example, a carboxyethyl group, a carboxypropyl group, a carboxybutyl group, a carboxypentyl group, an anion thereof (carboxylate), or an alkali metal salt thereof.
  • a group derived from a carboxylic acid represented by the general formula [2] such as a group of (for example, sodium salt, potassium salt, etc.) and a group of these organic ammonium salt (for example, trimethylammonium salt, triethylammonium salt, ammonium salt, etc.)
  • Groups of these organic ammonium salts (for example trimethyl)
  • a preferred combination of R 3 and R 4 and a preferred combination of R 5 and R 6 are any one (that is, any one of R 3 and R 4 and R 5 and Any one of R 6 ) is an alkyl group having 1 to 5 carbon atoms (for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, neopentyl group, etc.), and the other is, for example, a carboxyethyl group, a carboxypropyl group, a carboxybutyl group, a carboxypentyl group, an anion thereof (carboxy Rate), groups of these alkali metal salts (for example, sodium salts, potassium salts, etc.),
  • the alkyl group represented by R 7 to R 10 may be linear, branched or cyclic, and usually has 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms.
  • Examples of the alkynyl group represented by R 7 to R 10 usually include those having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms. Specific examples include ethynyl group, 2-propynyl group, 3-butynyl group, Examples include 1-methyl-2-propynyl group, 4-pentynyl group, 2-methyl-4-pentynyl group, 5-hexynyl group and the like.
  • Examples of the aryl group represented by R 7 to R 10 usually include those having 6 to 10 carbon atoms, and specific examples include a phenyl group and a naphthyl group.
  • the alkoxy group represented by R 7 to R 10 may be linear, branched or cyclic, and usually includes those having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms.
  • Examples of the aryloxy group represented by R 7 to R 10 usually include those having 6 to 10 carbon atoms, and specific examples include a phenyloxy group and a naphthoxy group.
  • alkylsulfonyl group represented by R 7 to R 10 examples include those in which the —OH group of the sulfo group (—SO 3 H) is substituted with an alkyl group, which may be linear, branched or cyclic.
  • those having 1 to 6 carbon atoms are mentioned, and specific examples include methylsulfonyl group, ethylsulfonyl group, n-propylsulfonyl group, isopropylsulfonyl group, n-butylsulfonyl group, isobutylsulfonyl group, sec- Butylsulfonyl group, tert-butylsulfonyl group, n-pentylsulfonyl group, isopentylsulfonyl group, sec-pentylsulfonyl group, tert-pentylsulfonyl group, neopentylsulfonyl group, n-hexylsulfonyl group, isohexylsulfonyl group, sec -Hexylsulfonyl group, tert-hexylsulfonyl
  • Examples of the arylsulfonyl group represented by R 7 to R 10 include those in which the —OH group of the sulfo group (—SO 3 H) is substituted with an aryl group, which may be linear, branched or cyclic. Usually, those having 6 to 10 carbon atoms are mentioned, and specific examples include phenylsulfonyl group, naphthylsulfonyl group and the like.
  • Examples of the halogen atom represented by R 7 to R 10 include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Examples of the substituted amino group represented by R 7 to R 10 include those in which 1 to 2 hydrogen atoms of the amino group are substituted with substituents.
  • substituents include alkyl groups, alkoxycarbonyl groups, acyl groups, and the like. Group, sulfo group and the like.
  • the alkyl group mentioned as a substituent of the substituted amino group represented by R 7 to R 10 may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms.
  • the alkoxycarbonyl group mentioned as a substituent of the substituted amino group represented by R 7 to R 10 may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms.
  • Examples of the acyl group exemplified as the substituent of the substituted amino group represented by R 7 to R 10 include those derived from aliphatic carboxylic acids and those derived from aromatic carboxylic acids.
  • the acyl group derived from the aliphatic carboxylic acid may be linear, branched or cyclic, and may further have a double bond in the chain, and usually has 2 to 20 carbon atoms, preferably carbon. Examples thereof include those having a number of 2 to 15, more preferably 2 to 10, and still more preferably 2 to 6.
  • acyl group derived from the aromatic carboxylic acid usually include those having 7 to 15 carbon atoms, preferably 7 to 11 carbon atoms, and specific examples thereof include a benzoyl group, a naphthoyl group, and an antoyl group.
  • R 7 ⁇ R 10 are, three of R 7 ⁇ R 10 are hydrogen atoms include those remaining one is a group derived from a sulfonic acid represented by the general formula [3], Among the groups derived from the sulfonic acid, for example, a sulfo group, an anion thereof (that is, a sulfonate), an alkali metal salt, an organic ammonium salt, and the like are preferable.
  • a sulfo group, a sulfonate, and an alkali metal salt thereof (for example, a sodium salt) ) And the like are more preferable, and those in which R 8 is a sulfonic acid-derived group represented by the general formula [3] are particularly preferable.
  • the alkyl group represented by R 11 may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 carbon atom.
  • alkynyl group represented by R 11 examples include those usually having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms. Specific examples include ethynyl group, 2-propynyl group, 3-butynyl group, 1-methyl group, and the like. -2-propynyl group, 4-pentynyl group, 2-methyl-4-pentynyl group, 5-hexynyl group and the like can be mentioned.
  • Examples of the aryl group represented by R 11 usually include those having 6 to 10 carbon atoms, and specific examples include a phenyl group and a naphthyl group.
  • an alkyl group is preferable, and particularly, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, and the like. More preferred.
  • n is usually an integer of 0 to 3, preferably 1 or 2, and more preferably 2.
  • the group derived from the sulfonic acid represented by the general formula [3] contained in the compound of the present invention increases the water-solubility between the dye molecules.
  • it is preferably introduced in a large amount, and is usually contained in the compound of the present invention in an amount of 1 to 4, preferably 3 to 4, and more preferably 4.
  • the sulfonic acid-derived group represented by the general formula [3] is contained in the present application compound, either one of R 3 and R 4 in the general formula [1] is used.
  • R 5 and R 6 and one of R 1 and R 2 which has a sulfonic acid-derived group represented by the general formula [3] as a substituent. It is preferably introduced as an alkyl group of ⁇ 5, and further introduced as a sulfonic acid-derived group represented by the general formula [3] in R 8 .
  • Examples of the compound represented by the general formula [1] include, for example, the general formula [1-1].
  • R 1a to R 6a are each independently an alkyl group having a group represented by the general formula [101] as a substituent, a group derived from a carboxylic acid represented by the general formula [2], or a general formula [3
  • R 8a represents a group derived from a sulfonic acid represented by the general formula [3]
  • R 11a represents an alkyl group.
  • n is the same as defined above, provided that at least one of R 1a to R 6a represents an alkyl group having a group represented by the general formula [101] as a substituent.
  • the alkyl group represented by R 1a to R 6a and having the group represented by the general formula [101] as a substituent is R 1 to R 1 in the general formula [1]. Examples thereof are the same as those exemplified for the alkyl group represented by R 6 and having the group represented by the general formula [101] as a substituent.
  • alkyl group that may have a substituent derived from a carboxylic acid-derived group represented by the general formula [2] or a sulfonic acid-derived group represented by the general formula [3] represented by R 1a to R 6a
  • the thing similar to the illustration of the alkyl group which may have is mentioned.
  • R 3a and R 4a are each independently an alkyl optionally having a carboxylic acid-derived group represented by the general formula [2] or a sulfonic acid-derived group represented by the general formula [3] as a substituent.
  • one of them is an alkyl group having a substituent derived from a carboxylic acid-derived group represented by the general formula [2] or a sulfonic acid group represented by the general formula [3], and the other is an alkyl group. What is group is preferable.
  • R 5a and R 6a are each independently an alkyl optionally having a carboxylic acid-derived group represented by the general formula [2] or a sulfonic acid-derived group represented by the general formula [3] as a substituent.
  • one of them is an alkyl group having a substituent derived from a carboxylic acid-derived group represented by the general formula [2] or a sulfonic acid group represented by the general formula [3], and the other is an alkyl group. What is group is preferable.
  • the alkyl group represented by R 11a include the same examples of the alkyl group represented by in R 11 in the general formula [1].
  • R 1a to R 6a is an alkyl group having a group represented by the general formula [101] as a substituent.
  • Preferred examples of the compound represented by the general formula [1-2] include those shown in Table 2 below.
  • Ph phenyl group (wherein R 17 represents an alkyl group or an aryl group, and R 1 to R 11 and n are the same as above)
  • the alkyl group represented by R 17 may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 3 carbon atoms.
  • Examples of the aryl group represented by R 17 usually include those having 6 to 10 carbon atoms, and specific examples include a phenyl group and a naphthyl group.
  • an indolenine compound represented by the general formula [8] indolenine skeleton portion
  • a compound represented by the general formula [9] [1 to 2 moles relative to the compound represented by the general formula [8]]
  • an acid anhydride represented by the general formula [10] [1 to 20 moles relative to the compound represented by the general formula [8] (for example, acetic anhydride, propionic anhydride, butyric anhydride, benzoic anhydride, etc.)
  • a solvent for example, carboxylic acids such as acetic acid, propionic acid, butyric acid, nitriles such as acetonitrile, propionitrile, n-butyronitrile, etc.
  • a solvent for example, carboxylic acids such as acetic acid, propionic acid, butyric acid, nitriles such as acetonitrile, propionitrile, n-butyronitrile, etc.
  • 0.1 to 24 hours preferably
  • the compound represented by the general formula [11] and the compound represented by the general formula [12] [pyrazole skeleton moiety) [0.5 to 10-fold mol, preferably 1 with respect to the compound represented by the general formula [11]
  • a basic catalyst for example, pyridine, triethylamine, N, N-dimethylaniline, piperidine, pyridine, 4-dimethylaminopyridine, 1,5-diazabicyclo [4.3.0] nona-5
  • Organic amines such as -ene, 1,8-diazabicyclo [5.4.0] undec-7-ene, tri-n-butylamine, metal hydrides such as sodium hydride, basic such as n-butyllithium
  • dehydrating condensing agents for example, inorganic dehydrating agents such as concentrated sulfuric acid, diphosphorus pentoxide, anhydrous zinc chloride, such as dicyclohexylcarbod
  • Nitriles such as acetonitrile, propionitrile, n-butyronitrile, etc., methanol, ethanol , Alcohols such as propanol, isopropanol, butanol, ethylene glycol, 1,4-butanediol, ethers such as tetrahydrofuran, dioxane, anisole, ethylene glycol monoethyl ether, sulfoxides such as dimethyl sulfoxide, etc. In which the reaction is carried out at 0 to 150 ° C. (preferably 40 to 120 ° C.) for 0.1 to 24 hours (preferably 0.5 to 12, more preferably 1 to 8 hours). The compound represented by [1] is obtained.
  • the method for introducing the group represented by the general formula [101] according to the present invention is the compound represented by the general formula [24] among the compounds represented by the general formula [1] [that is, the general formula In the case of synthesizing a compound in which R 1 in [1] is a group represented by the general formula [101] (provided that this is an alkyl group having R 104 as a substituent). An example is described below.
  • T 12 represents an alkylene group
  • A represents tetrafluoroborate or hexafluorophosphate
  • R 3 to R 12 , R 101 to R 103 , m and n are the same as above
  • an alkyl group having a group derived from a carboxylic acid R 1 is represented by the general formula [2] as a substituent (i.e. , -T 12 -COOR 12 group).
  • the alkylene group represented by T 12 may be linear or branched, preferably linear, and usually has 1 to 6 carbon atoms.
  • it is a group of 1-4, and specifically, for example, a linear alkylene group such as a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, such as an ethylidene group, a propylene group Isopropylidene group, ethylethylene group, 1,2-dimethylethylene group, 1,2-diethylethylene group, 1,2-di-n-propylethylene group, 1,2-di-n-butylethylene group, etc.
  • a branched alkylene group and the like can be mentioned, among which a linear alkylene group is preferable, and an ethylene group, a pentamethylene group and the like are more preferable.
  • a compound represented by the general formula [22] is made basic with a succinimidation reagent such as a compound represented by the general formula [62] (1 to 10-fold mol relative to the compound represented by the general formula [22]).
  • Catalysts eg N-ethyldiisopropylamine, pyridine, triethylamine, N, N-dimethylaniline, piperidine, 4-dimethylaminopyridine, 1,5-diazabicyclo [4.3.0] non-5-ene, 1,8-diazabicyclo [ 5.4.0] in the presence of undec-7-ene, organic amines such as tri-n-butylamine, etc.
  • an appropriate solvent for example, amides such as DMF, DMA, acetamide, N-methylpyrrolidone, etc.
  • a compound represented by the general formula [23] and a cationizing reagent represented by, for example, the general formula [102] (1 to 5 moles relative to the compound represented by the general formula [23]) are mixed with an appropriate solvent (for example, The compound represented by the general formula [24] can be obtained by reacting at 0 to 40 ° C. for 0.1 to 12 hours in amides such as DMF, DMA, acetamide and N-methylpyrrolidone).
  • the succinimidation reagent according to the present invention is not limited to the compound represented by the above general formula [62], but includes all those usually used in this field. Specifically, for example, di (N-succinimidyl) carbonate ( DSC), 2-succinimide-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), 2- (5-norbornene-2,3-dicarboximide) -1,1,3,3- Examples include tetramethyluronium tetrafluoroborate (TNTU).
  • DSC di (N-succinimidyl) carbonate
  • TSTU 2-succinimide-1,1,3,3-tetramethyluronium tetrafluoroborate
  • TNTU tetramethyluronium tetrafluoroborate
  • a basic catalyst for example, triethylamine, N-ethyldiisopropylamine, pyridine, N, N-dimethylaniline, piperidine, 4-dimethylaminopyridine, 1,5-diazabicyclo [4.3. 0] nona-5-ene, 1,8-diazabicyclo [5.4.0] undec-7-ene, organic amines such as tri-n-butylamine, and the like.
  • a basic catalyst for example, triethylamine, N-ethyldiisopropylamine, pyridine, N, N-dimethylaniline, piperidine, 4-dimethylaminopyridine, 1,5-diazabicyclo [4.3. 0] nona-5-ene, 1,8-diazabicyclo [5.4.0] undec-7-ene, organic amines such as tri-n-butylamine, and the like.
  • Ts tosyl (p-toluenesulfonyl) group (wherein X represents a halogen atom, R 1 , R 3 , R 4 and R 7 to R 10 are the same as above)
  • examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • the ketone compound represented by the general formula [13] and the compound represented by the general formula [14] are mixed in a suitable solvent (for example, carboxylic acids such as acetic acid and propionic acid, such as ethylene glycol and 1,4-butanediol). Alcohols, etc.) are reacted at 40 to 250 ° C. for 0.1 to 24 hours to obtain a compound represented by the general formula [21] (for example, JournalJof Organic Chemistry, 42 (14), 2474-80, 1977). etc).
  • a suitable solvent for example, carboxylic acids such as acetic acid and propionic acid, such as ethylene glycol and 1,4-butanediol.
  • Alcohols, etc. are reacted at 40 to 250 ° C. for 0.1 to 24 hours to obtain a compound represented by the general formula [21] (for example, JournalJof Organic Chemistry, 42 (14), 2474-80, 1977). etc).
  • the ketone compound represented by the general formula [13] is a commercially available product (for example, 3-methyl-2-butanone, 3-methyl-2-pentanone, 3-methyl-2-hexanone, 1-cyclopropylethanone, 1-cyclohexane). Butylethanone etc.) may be used, or those appropriately synthesized by conventional methods may be used. Examples of the synthesis of the ketone compound include, for example, a compound having an ethyl 2-methylacetoacetate and a leaving group (for example, a halogen atom, a tosylate group, etc.) as a basic catalyst (for example, metal hydrides such as sodium hydride and potassium hydride).
  • Alkali metal carbonates such as lithium carbonate, sodium carbonate and potassium carbonate, alkali metal alkoxides such as sodium methoxide, sodium ethoxide and potassium tert-butoxide, and basic alkali metal compounds such as n-butyllithium
  • a suitable solvent eg DMF, DMA, acetamide, N-methylpyrrolidone etc. amides such as methanol, ethanol, propanol, isopropanol, butanol, ethylene).
  • the diketone compound represented by the general formula [18] and hydrazine are mixed in an appropriate solvent (for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, etc.) at 60 to 100 ° C. for 1 to 4 hours.
  • an appropriate solvent for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, etc.
  • the 4H-pyrazole compound represented by the general formula [19] is combined with the halogenated compound represented by the general formula [20] or the tosylate compound represented by the general formula [21] and an appropriate solvent (for example, chlorobenzene, 1,2 -Halogenated aromatic hydrocarbons such as dichlorobenzene, halogenated hydrocarbons such as 1,2-dichloroethane, aromatic hydrocarbons such as toluene, xylene and benzene, such as DMF, DMA, acetamide, N- N-alkylation reaction in amides such as methylpyrrolidone at 80 to 140 ° C. for 1 to 12 hours to obtain a compound represented by the general formula [12] (for example, J. Chem. Soc., Perkin Trans .1. 947 ⁇ 952. 1984 etc.).
  • an appropriate solvent for example, chlorobenzene, 1,2 -Halogenated aromatic hydrocarbons such as dichlorobenzene, halogenated hydrocarbon
  • diketone compound represented by the general formula [18] commercially available products (for example, 3,3-dimethyl-2,4-pentanedione, etc.) may be used, or those synthesized appropriately by a conventional method may be used.
  • examples of the synthesis of the ketone compound include, for example, a compound having a leaving group (for example, a halogen atom, a tosylate group, etc.) having 3-methyl-2,4-pentanedione or 4-acetyl-5-oxohexanoic acid ethyl ester as a base.
  • metal hydrides such as sodium hydride and potassium hydride, alkali metal carbonates such as lithium carbonate, sodium carbonate and potassium carbonate, alkalis such as sodium methoxide, sodium ethoxide and potassium tert-butoxide
  • a metal alkoxide for example, a basic alkali metal compound such as n-butyllithium, for example, an alkali metal amide such as lithium diisopropylamide, etc.
  • an appropriate solvent for example, DMF, DMA, acetamide, N-methylpyrrolidone, etc.
  • Amides such as methanol, ethanol, propanol, In alcohols such as propanol, butanol and ethylene glycol, ethers such as tetrahydrofuran, dioxane and ethylene glycol monoethyl ether, and sulfoxides such as dimethyl sulfoxide
  • ethers such as tetrahydrofuran, dioxane and
  • the compound represented by the general formula [1] of the present invention thus obtained (hereinafter sometimes abbreviated as “the compound [1] of the present invention”).
  • the compound [1] of the present invention Is a fluorescent substance having an excitation wavelength near 635 nm, and is expected to be used as a fluorescent labeling substance, an internal standard substance in electrophoresis, and the like.
  • the compound [1] of the present invention has a group represented by the general formula [101] (that is, an ammonium cation group) in its molecule, when this is electrophoresed from plus to minus, The presence of the ammonium cation group according to the invention makes it possible to delay the migration time. Furthermore, in the compound of the present invention, a group derived from a carboxylic acid represented by the general formula [2] or a group derived from a sulfonic acid represented by the general formula [3] can be introduced as an anion group. Since the number of cation groups and anion groups in the molecule can be appropriately controlled, it becomes possible to synthesize the compound of the present invention in which the mobility during electrophoresis is controlled.
  • the compound [1] of the present invention has the same basic skeleton, and the group represented by the general formula [101] according to the present invention is a group derived from the carboxylic acid represented by the general formula [2] or the general formula [2].
  • the anion group-containing compound according to the present invention is When this is electrophoresed from plus to minus, the migration time can be made faster than that of the compound [1] according to the present invention, so that the peak of the substance to be measured is between the peaks of the two internal standard substances. Thus, the peak of the substance to be measured can be easily identified.
  • anion group-containing compound according to the present invention include, for example, a compound represented by the general formula [50] or a salt thereof
  • R 1b to R 6b each independently has an amide bond, and is represented by the general formula [2]
  • R 13 represents an alkyl group optionally having a sulfonic acid-derived group as a substituent
  • R 7b to R 10b are each independently an alkyl group or an alkynyl group.
  • R 11b represents a hydrogen atom, an alkyl group, an alkynyl group or an aryl group
  • n ′ represents an integer of 0 to 3.
  • R 1b to R 6b is a group derived from a carboxylic acid represented by the general formula [2] or a group derived from a sulfonic acid represented by the general formula [3], or these as substituents Have. ].
  • the compound represented by the general formula [50] or a salt thereof is a fluorescent substance having an excitation wavelength near 635 nm, and is a particularly useful internal standard substance in measurement by fluorescence detection.
  • the alkyl group which may have a group derived from as a substituent it may have an amide bond represented by R 1 to R 6 in the general formula [1].
  • the same thing as the illustration of the alkyl group which may have as a substituent the group derived from carboxylic acid shown by or the group derived from the sulfonic acid shown by General formula [3] is mentioned.
  • the alkyl group having, a part of the hydrogen atoms in the alkyl group which may have an amide bond may be the substituent (a group derived from the carboxylic acid represented by the general formula [2] or the general formula [3] And those substituted with the carbon atom at the terminal of the alkyl group are preferred.
  • alkyl group, alkynyl group, aryl group, alkoxy group, aryloxy group, alkylsulfonyl group, arylsulfonyl group, halogen atom and substituted amino group represented by R 7b to R 10b are represented by R 7 in the general formula [1].
  • alkyl group represented by ⁇ R 10 alkynyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylsulfonyl group, an arylsulfonyl group, and examples and respective same halogen atom and a substituted amino group.
  • Examples of the alkyl group, alkynyl group and aryl group represented by R 11b in the general formula [50] are the same as those of the alkyl group, alkynyl group and aryl group represented by R 11 in the general formula [1]. Can be mentioned.
  • an alkyl group is preferable, and a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, and the like are more preferable.
  • n ′ is usually an integer of 0 to 3, preferably 1 or 2, and more preferably 2.
  • the compound represented by the general formula [50] which is an anion group-containing compound according to the present invention can be synthesized, for example, according to a conventional method (International Publication WO2007 / 114398).
  • the measurement target substances that can use the compound of the present invention as an internal standard substance or a fluorescent labeling substance include all substances that are usually measured in this field, and are not particularly limited.
  • Chain eg C-peptide, angiotensin I, etc.
  • protein eg immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), ⁇ 2-microglobulin, albumin, degradation products thereof, serum proteins such as ferritin, such as amylase, alkaline phosphatase, ⁇ -glutamyltransferase, and other enzyme proteins such as tuberculosis, pneumococci, diphtheria, meningococcus, gonococci, Staphylococcus, Streptococcus, Enterobacteriaceae, Escherichia coli, Helicobacter pylori, etc.
  • Viruses such as bacteria, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, such as Candida, cryptococcus, etc.
  • Proteins, peptides or sugar chains derived from microorganisms such as spirochetes such as fungi, leptospira, syphilis treponema, chlamydia, mycoplasma, etc.
  • Allergens derived from pollen such as blue-winged frog, hurghaya, rye, etc., animals such as cats, dogs, crabs, foods such as rice, egg white, fungi, insects, wood, drugs, chemicals, etc.
  • lipids such as lipoproteins, proteases such as trypsin, plasmin, serine proteases, such as AFP, PSA, CEA, PGI , Tumor marker proteins such as PGII, etc.] sugar chains (for example, CA19-9, PIVKA-II, CA125, tumor markers sugar chain antigen sugar chains such as sugar chains possessed by substances having special sugar chains produced by cancer cells, For example, ABO sugar chain), lectins (for example, concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin, etc.), phospholipids (for example, cardiopyrine), lipopolysaccharides (for example, endotoxin), chemical substances (for example, PTH, T3, T4, TSH, insulin, Hormones such as H, FSH, and prolactin, such as tribut
  • tumor marker proteins such as AFP, PSA, CEA, PGI, PGII, CA19-9, PIVKA-II, CA125
  • Tumor marker sugar chain antigen sugar chains such as sugar chains possessed by substances having special sugar chains produced by cancer cells are preferred, and AFP and PIVKA-II are particularly preferred.
  • Electrophoresis in the measurement method using the compound of the present invention as an internal standard substance or a fluorescent labeling substance includes all the methods usually used in this field, but in particular, electrophoresis performed in a capillary (capillary)
  • the electrophoresis method is more preferable.
  • an electrophoresis method performed with a capillary chip is preferable.
  • Capillary (micro) chip electrophoresis is a technique in which a capillary with a cross-section diameter of 100 ⁇ m or less is formed on a chip substrate, and electrophoresis is performed in this capillary. It is present in a sample by applying a voltage to the capillary. This is a method of separating the difference in the charge of the substance to be used as the difference in mobility.
  • Capillary electrophoresis is classified into capillary zone electrophoresis and capillary gel electrophoresis depending on the electrophoresis solution used, but the method of the present invention can be applied to any of them. Considering the accuracy of separation, among the above, capillary gel electrophoresis is preferable.
  • the material of the capillary used in the capillary electrophoresis is not particularly limited as long as it is usually used in this field.
  • a silica-based compound such as glass, quartz, silicon, etc.
  • Examples include synthetic polymers such as click olefin copolymer (COC), cyclic olefin polymer (COP), polymethyl methacrylate, polymethyl siloxane, polyvinyl chloride, polyurethane, polystyrene, polysulfone, polycarbonate, and polytetrafluoroethylene. Is preferred.
  • the inner diameter and length of the capillary are not particularly limited as long as they can separate the substance to be measured, but the inner diameter is usually 1 to 1000 ⁇ m, preferably 1 to 200 ⁇ m, more preferably 1 to 100 ⁇ m.
  • the length is usually 0.1 mm to 100 cm, preferably 0.1 mm to 20 cm, more preferably 0.1 mm to 10 cm.
  • the electrophoresis solution used in the electrophoresis method according to the present invention is not particularly limited as long as it is usually used in this field.
  • the buffer include lactate buffer, citrate buffer, acetate buffer, succinate buffer, glycine buffer, phthalate buffer, Examples include phosphate buffer, triethanolamine buffer, borate buffer, glycine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer, tartaric acid buffer, borate buffer, etc. However, it is not limited to these.
  • polyethers such as polyethylene oxide (polyethylene glycol) and polypropylene oxide
  • polyalkyleneimines such as polyethyleneimine
  • polyacrylic acid polyacrylic acid ester
  • polymethyl acrylate polymethyl acrylate
  • Polyacrylic acid polymers such as polyamide polymers such as polyacrylamide and polymethacrylamide, such as polymethacrylic acid polymers such as polymethacrylic acid, polymethacrylate, polymethyl methacrylate, such as polyvinyl acetate, polyvinylpyrrolidone, polyvinyl Polyvinyl polymers such as oxazolidone, such as water-soluble hydroxyl polymers such as pullulan, erucinane, xanthan, dextran, guar gum, etc.
  • Buffers used as electrophoresis solutions for capillary zone electrophoresis using water-soluble cellulose compounds such as cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, derivatives thereof, and polymers such as copolymers containing multiple types of monomer units constituting these polymers The thing etc. which were added to the liquid are mentioned. In addition, you may add the said polymer in combination of 2 or more types.
  • the molecular weight of the polymer as described above is usually 500 Da to 6000 kDa, preferably 1 to 1000 kDa, more preferably 100 to 1000 kDa.
  • the concentration of the polymer may be appropriately selected from the range usually used in this field, and is usually 0.01 to 40% (W / V), preferably 0.01 to 20% (W / V), more preferably 0.1 to 10% (W / V).
  • the viscosity of the electrophoresis buffer when the above filler is added to the electrophoresis buffer is usually 2 to 1000 centipoise, preferably 5 to 200 centipoise, more preferably 10 to 100 centipoise.
  • the electrophoresis solution may contain substances for reducing the influence of electroosmotic flow, such as polyacrylamide, polyethylene glycol, polyethyleneimine, fluorine-containing aromatic hydrocarbons, saccharides, etc., and the concentration thereof is usually in this field. What is necessary is just to set in the range used.
  • the voltage at the time of electrophoresis in the electrophoresis method according to the present invention varies depending on the electrophoresis solution, the apparatus used, and the like, it may be appropriately set from the range normally used in this field.
  • the method for introducing the compound [1] of the present invention which is a sample or internal standard substance in the electrophoresis method according to the present invention, and the anion group-containing compound according to the present invention may be a method usually used in this field, and is particularly limited. However, there are, for example, a suction method, a pressurizing method, an electrical introduction method and the like, and among them, the pressurizing method is preferable.
  • the internal standard substance according to the present invention may be introduced in a state of being dissolved in advance in the sample, or a solution containing the sample and the internal standard substance may be separately introduced.
  • the amount of the internal standard substance to be introduced varies depending on conditions such as the inner diameter and length of the capillary, the type of detector and the sensitivity, but usually the same amount as the sample injection amount is used.
  • the sample or the internal standard substance according to the present invention may be introduced by concentrating by known isotachophoresis (ITP) and then introducing the concentrate as it is into capillary electrophoresis. In this case, for example, when microchip electrophoresis is performed, it is desirable to perform ITP electrophoresis and capillary electrophoresis continuously using a chip in which ITP electrophoresis and capillary electrophoresis are connected.
  • the sample may be a solution containing the substance to be measured, and is not particularly limited.
  • body fluid such as serum, plasma, spinal fluid, synovial fluid, lymph fluid, urine, feces, etc.
  • Cleaning liquid after cleaning biological samples such as excrement, sputum, pus, skin-derived material, such as food, beverages, tap water, seawater, lake water, river water, factory waste liquid, semiconductor cleaning water, medical equipment, etc.
  • Environmental samples such as water, etc., and re-dissolve them in water or in buffers such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc.
  • the processed material etc. which were obtained by comprising are mentioned.
  • the electrophoresis method according to the present invention is performed, for example, on a capillary having an inner diameter of 50 to 100 ⁇ m and a length of 1 to 10 cm, for example, 0.1 to 1.0% polydimethylaminoethyl methacrylate, 1 to 5% glycerol, 0.01 to 0.1%.
  • a Tris-HCl buffer containing BSA is filled, and a sample containing one internal standard is introduced into the capillary from the starting end of the capillary by a pressure method of 1 to 10 psi for 30 to 60 seconds, then 30 to 1 to 10 psi.
  • electrophoresis is performed by applying a voltage of 1000 to 3000 V for 10 to 60 minutes, and a detector such as a fluorescence detector or a UV detector Measured by
  • electrophoresis is performed under the conditions as described above, and the electrophorogram is obtained by measuring the migration state of the measurement target substance and the internal standard substance with a detector such as a fluorescence detector or a UV detector. After identifying the peak of the internal standard substance in the electropherogram, the peak of the measurement target substance is identified from the migration time and the like.
  • the internal standard substance and the standard standard of the measurement target substance are electrophoresed in advance, and the two internal standard substances and the measurement target are measured.
  • the migration time of the substance, the ratio of the migration time of each internal standard substance, and the ratio of the migration time of the internal standard substance and the measurement target substance are measured.
  • the sample and the substance to be measured are electrophoresed under the same conditions, and the peaks of the two internal standard substances are specified from the migration time and the ratio of the migration times of the internal standard substances obtained previously.
  • the peak of the measurement target substance is identified from the migration time of the two internal standard substances and the ratio of the migration time of the internal standard substance and the measurement target substance determined previously.
  • the compound of the present invention can also be used as a labeling substance.
  • a reactive group capable of binding to the labeling substance may be introduced into the compound of the present invention.
  • the methods usually used in this field can be mentioned.
  • Example 2 Measurement of fluorescence intensity of the compound of the present invention The relative fluorescence intensity of the known compound (13) and the compound (15) of the present invention was estimated. First, the fluorescence intensity per 1 ⁇ M was measured from the fluorescence intensity of the known compound (13), and the relative fluorescence intensity of the compound of the present invention was estimated when this fluorescence intensity value was 100.
  • the known compound (13) was dissolved in 1 mL of purified water. This solution was diluted 200 times with 50 mM phosphate buffer (pH 7.5), and the OD value was measured.
  • the compound (15) of the present invention obtained in (1) of Example 1 was dissolved in 1 mL of purified water, and 50 mM phosphate buffer (pH 7. Diluted in 5). At this time, assuming that the fluorescence intensity per unit OD value of the known compound (13) was 100, the relative fluorescence intensity of the following compound (15) of the present invention was estimated. The results are shown in Table 2.
  • AFP separation measurement was performed using the compound of the present invention as an internal standard substance in electrophoresis.
  • AFP ⁇ -fetoprotein
  • a capillary chip having the layout shown in FIG. 2 was prepared as follows according to the method described in “Technology and Application of Micro Chemical Chips Takehiko Kitamori et al. 2004 (Maruzen Co., Ltd.)”. That is, a photoresist film was formed on Si formed on a quartz substrate. This photoresist was exposed and developed using a mask having a capillary design (layout) shown in FIG. After removing the portion of the Si where the photoresist was removed by development by sputtering, wet etching was performed using a hydrogen fluoride solution to produce capillary channel grooves (capillaries) in the quartz substrate. After removing the photoresist and the Si film remaining on the quartz substrate, the quartz substrate and a cover plate having a well (well) for reservoir were bonded together by an HF bonding method to produce a capillary chip.
  • TB is a well for introducing a trailing buffer
  • LB1 and LB2 are wells for introducing a reading buffer
  • S is a well for introducing a sample for electrophoresis
  • R1 is a well for introducing a test solution (250 bp DNA labeled antibody-containing solution).
  • W1, W2, and W3 represent drain wells, respectively.
  • Reading buffer Polydimethylacrylamide (pDMA) 0.6% (w / v), glycerol 3% (w / v), NaCl 75mM, bovine serum albumin (BSA) 0.01% and LCA 4 mg / ml
  • BSA bovine serum albumin
  • the reaction solution was allowed to stand on ice and allowed to undergo antigen-antibody reaction for about 30 minutes to form a fluorescently labeled antibody-AFP immune complex.
  • the final concentration of the fluorescently labeled antibody is 100 nM.
  • the obtained immune complex-containing reaction solution was used as a sample for electrophoresis.
  • Electrophoresis procedure a) Introduction of electrophoresis sample and test solution 10 ⁇ L of electrophoresis sample (fluorescent-labeled antibody-AFP immune complex-containing solution) in the S well (electrophoresis sample introduction well) of FIG.
  • FIG. 3 schematically shows the positional relationship between the sample for electrophoresis and the sample solution in the capillary.
  • the shaded portion indicates the placement portion of the electrophoresis sample
  • the dot portion indicates the placement portion of the reagent solution.
  • the 250 bp DNA labeled antibody in the test solution is fluorescent labeled antibody in the sample for electrophoresis.
  • ITP isotachophoresis
  • CGE Capillary gel electrophoresis
  • FIG. 4 shows an electrophoretic image (electrophoresis chromatogram) when a sample for electrophoresis is used.
  • the vertical axis indicates the fluorescence intensity
  • the horizontal axis indicates the retention time.
  • An anti-prothrombin antibody is prepared and processed by a conventional method to obtain an anti-prothrombin antibody Fab ′ fragment.
  • a fluorescent substance HiLyte647 (manufactured by AnaSpec) is introduced into the amino group of the fragment by a conventional method, and a HiLyte647-labeled anti-prothrombin antibody Fab ′ fragment is prepared. Fragments (fluorescently labeled antibodies) were prepared.
  • the reaction solution was allowed to stand on ice and allowed to react for about 30 minutes with an antigen-antibody to form a fluorescently labeled antibody-PIVKA II immune complex.
  • the final concentration of the fluorescently labeled antibody is 100 nM.
  • the obtained immune complex-containing reaction solution was used as a sample for electrophoresis.
  • Reagent 250bp DNA labeled antibody-containing solution
  • a leading buffer containing 50 mM Cl - ion content
  • Electrophoretic procedure In the same manner as in Experimental Example 1, the electrophoresis sample and sample solution were introduced, concentrated with ITP, CGE was performed, and the fluorescence intensity of the capillary part 2 cm from the LB2 channel cross part was fluorescent by 635 nm laser excitation. The measurement was made over time with a microscope (BX-50; manufactured by KS Olympus Corporation).
  • FIG. 5 shows an electrophoretic image (electrophoresis electropherogram) when a sample for electrophoresis is used.
  • the vertical axis represents fluorescence intensity
  • the horizontal axis represents retention time.

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Abstract

La présente invention concerne un composé représenté par la formule générale [1] ou un sel de celui-ci, qui est un nouveau dérivé du colorant cyanine ayant une intensité de fluorescence élevée dans une région de longueurs d'onde courtes. Le dérivé du colorant cyanine a une structure comprenant un squelette pyrazole et un squelette indole combiné à celui-ci par le biais d'une chaîne polyméthine, et a été rendu contrôlable en ce qui concerne sa mobilité électrophorétique en introduisant de plus un cation ammonium quaternaire à l'intérieur d'une molécule ayant cette structure. (Dans la formule [1], les R1 à R6 représentent chacun indépendamment, par exemple, un alkyle ayant un substituant représenté par la formule générale [101], les R7 à R10 représentent chacun indépendamment un atome d'hydrogène, etc., R11 représente un atome d'hydrogène, un alkyle, etc., et n représente un entier de 0 à 3, au moins un des R1 à R6 représentant un alkyle ayant un substituant représenté par la formule générale [101]).
PCT/JP2010/057467 2009-04-28 2010-04-27 Colorant cyanine à base de pyrazole contenant un cation ammonium quaternaire Ceased WO2010126045A1 (fr)

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US13/318,089 US8546586B2 (en) 2009-04-28 2010-04-27 Pyrazole-based cyanine dye containing quaternary ammonium cation
ES10769743.5T ES2508340T3 (es) 2009-04-28 2010-04-27 Colorante de cianina a base de pirazol que contiene catión de amonio cuaternario
EP10769743.5A EP2426123B1 (fr) 2009-04-28 2010-04-27 Colorant cyanine à base de pyrazole contenant un cation ammonium quaternaire
JP2011511413A JP5598468B2 (ja) 2009-04-28 2010-04-27 第４級アンモニウムカチオン含有ピラゾール系シアニン色素

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5268486A (en) 1986-04-18 1993-12-07 Carnegie-Mellon Unversity Method for labeling and detecting materials employing arylsulfonate cyanine dyes
US5571388A (en) 1984-03-29 1996-11-05 Li-Cor, Inc. Sequencing near infrared and infrared fluorescense labeled DNA for detecting using laser diodes and suitable labels thereof
WO1998015829A1 (fr) * 1996-10-07 1998-04-16 Amersham International Plc Analyse de glucides
WO1999005221A1 (fr) * 1997-07-28 1999-02-04 Nycomed Amersham Plc Colorants de cyanine
WO2001002374A1 (fr) 1999-07-06 2001-01-11 Surromed, Inc. Colorants fluorescents pontes, leur preparation et leur utilisation dans des dosages
JP2003034696A (ja) 2001-07-19 2003-02-07 Fuji Photo Film Co Ltd 蛍光ヌクレオチド及びそれを用いた標識法
JP2003034697A (ja) 2001-07-19 2003-02-07 Fuji Photo Film Co Ltd 蛍光ヌクレオチド及びそれを用いた標識法
WO2007114398A1 (fr) 2006-03-31 2007-10-11 Wako Pure Chemical Industries, Ltd. Colorant de cyanine de type pyrazole

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2331444A1 (de) * 1973-06-20 1975-01-23 Hoechst Ag Neue styryl-benzoxazole, verfahren zu deren herstellung und ihre verwendung als optische aufhellungsmittel
JPH01246286A (ja) * 1988-03-28 1989-10-02 Hamari Yakuhin Kogyo Kk ポルフィリン誘導体
GB0615211D0 (en) * 2006-07-31 2006-09-06 Ge Healthcare Uk Ltd Asymmetric flouro-substituted polymethine dyes

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571388A (en) 1984-03-29 1996-11-05 Li-Cor, Inc. Sequencing near infrared and infrared fluorescense labeled DNA for detecting using laser diodes and suitable labels thereof
US5800995A (en) 1984-03-29 1998-09-01 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
US5268486A (en) 1986-04-18 1993-12-07 Carnegie-Mellon Unversity Method for labeling and detecting materials employing arylsulfonate cyanine dyes
US5486616A (en) 1986-04-18 1996-01-23 Carnegie Mellon University Method for labeling and detecting materials employing arylsulfonate cyanine dyes
US5569766A (en) 1986-04-18 1996-10-29 Carnegie Mellon University Method for labeling and detecting materials employing arylsulfonate cyanine dyes
WO1998015829A1 (fr) * 1996-10-07 1998-04-16 Amersham International Plc Analyse de glucides
WO1999005221A1 (fr) * 1997-07-28 1999-02-04 Nycomed Amersham Plc Colorants de cyanine
WO2001002374A1 (fr) 1999-07-06 2001-01-11 Surromed, Inc. Colorants fluorescents pontes, leur preparation et leur utilisation dans des dosages
JP2003034696A (ja) 2001-07-19 2003-02-07 Fuji Photo Film Co Ltd 蛍光ヌクレオチド及びそれを用いた標識法
JP2003034697A (ja) 2001-07-19 2003-02-07 Fuji Photo Film Co Ltd 蛍光ヌクレオチド及びそれを用いた標識法
WO2007114398A1 (fr) 2006-03-31 2007-10-11 Wako Pure Chemical Industries, Ltd. Colorant de cyanine de type pyrazole

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Modern Synthetic Reactions", 1972, pages: 492,510,
ADV. HETEROCYCLE. CHEM., vol. 34, 1983, pages 53 - 78
J. CHEM. SOC. , PERKIN TRANS., vol. 1, 1984, pages 947 - 952
JOURNAL OF ORGANIC CHEMISTRY, vol. 42, no. 14, 1977, pages 2474 - 80
POSER JW; PRICE PA, J. BIOL. CHEM., vol. 254, no. 2, 25 January 1979 (1979-01-25), pages 431 - 6
See also references of EP2426123A4 *
TAKEHIKO KITAMORI ET AL.: "Technology and applications of micro chemical chip", 2004, MARUZEN CO., LTD.

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EP2426123A4 (fr) 2012-10-31
US20120046474A1 (en) 2012-02-23
US8546586B2 (en) 2013-10-01
JP5598468B2 (ja) 2014-10-01
EP2426123B1 (fr) 2014-08-06
ES2508340T3 (es) 2014-10-16
EP2426123A1 (fr) 2012-03-07
JPWO2010126045A1 (ja) 2012-11-01

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